Structure and mechanism studies of sugar epimerase from dictyoglomus turgidum

Structure determination of Malonyl CoA-acyl carrier protein transacylase XoMCAT .... 11 Figure 1.5Work flow for solving the protein structure by X-ray crystallography ...15 Figure1.6Sche

Dissertation for Degree of Doctor

Supervisor: Prof Lin-Woo Kang

Structure and Mechanism Studies of

Department of Advanced Technology Fusion

Graduate School of Konkuk University

Structure and Mechanism Studies of

Sugar Epimerase

fromDictyoglomus turgidum

A Dissertationsubmitted to the Department of Advanced Technology Fusion and the Graduate School of Konkuk University in partial fulfillment of the requirements for the

degree of Doctor of Philosophy

Submitted by

PHAM TANVIET

April, 2014

This certifies that the Dissertation of PHAM TAN VIET is approved.

Approved by Examination Committee:

TABLE OF CONTENTS

List of Tables v

List of Figures vi

Abstract ix

Chapter 1 Structure and mechanism studies of sugar epimerase from Dictyoglomus turgidum 1

1.1 Introduction 1

1.1.1 The important roles of carbohydrate in life 1

1.1.2 Epimerization reaction mechanism 4

1.1.3 Three-dimensional structure determination methods 10

1.1.3.1 Electron microscopy 10

1.1.3.2 Atomic force microscopy 12

1.1.3.3 Nuclear magnetic resonance (NMR) 13

1.1.3.4 X-ray crystallography 14

1.1.3.4.1 Protein crystallization principle 16

1.1.3.4.2 Vapor diffusion methods for protein crystallization 18

1.2 Methods 20

1.2.1 Cloning of cellobiose 2-epimerase 20

1.2.2 Overexpression and Purification of cellobiose 2-epimerase 23

1.2.3 Crystallization and X-ray data collection 25

1.2.4 Structure determination and refinement 29

1.3 Results and Discussions 31

1.3.1 Quality of the refined model 31

1.3.2 Overal structure of DT_epimerase 34

1.3.3 Epi_DT complex structure 40

Chapter 2 Structure determination of Malonyl CoA-acyl carrier protein transacylase (XoMCAT) 55

2.1 Introduction 55

2.2 Methods 56

2.2.1 Crystallization and X-ray data collection 56

2.2.2 Structure determination and refinement 58

2.3 Results and Discussion 58

2.3.1 Overall structure of XoMCAT 58

2.3.2 Active site structure of XoMCAT 62

2.3.3 Proposed catalytic mechanism of XoMCAT 63

2.3.4 Computational protein-protein docking of XoMCAT and ScACP 64

Chapter 3 Structure determination of Cytochrome P450 107W1 (CYP107W1) 68

3.1 Introduction 68

3.2 Methods 72

3.2.1 Crystallization and X-ray data collection 72

2.2.3 Structure determination and refinement 73

3.3 Results and Discussion 74

3.3.1 Quality of the refined model 74

3.3.2 Overall structure of CYP107W1 77

3.3.3 CYP107W1 complex structure 81

References 84

Supplemental crystallization data 103

Abstract (in Korean) 137

Acknowledgment 139

Publications 141

List of Tables

Table 1.1Data collection statistics of Epi_DT 33

Table 1.2Interactions between lactose and Epi_DT 47

Table 2.1Datacollection statistics of XoMCAT 57

Table 3.1Datacollection statisticsof CYP107W1 75

Table S.1Data collection statistics of CjPDF 105

Table S.2Data collection statistics of AbALR 110

Table S.3Datacollection statistics of ADH 114

Table S.4Datacollection statistics of XometB 118

Table S.5Datacollection statistics of SFC-1 122

Table S.6Data collection statistics of XoGroEL 126

Table S.7Datacollection statistics of XoGroES 131

Table S.8Datacollection statistics of AbDdl 135

List of Figures

Figure 1.1Major pathways in carbohydrate metabolism 2

Figure 1.2Epimerase mechanism in general 6

Figure1.3Proposed catalytic mechanisms for AGE and YihS 8

Figure1.4Schematic drawing of electron microscopy apparatus 11

Figure 1.5Work flow for solving the protein structure by X-ray crystallography 15

Figure1.6Schematic illustration of a protein crystallization phase diagram 17

Figure1.7Crystalsin the reservoir solution 19

Figure1.8pET-29b-His-Tev vector sequence 22

Figure1.9Purified His-tagged Epi_DT and untagged Epi_DT 24

Figure1.10Crystals of His-tagged and untagged Epi_DT 28

Figure1.11Diffraction images of His-tagged (a) and untagged (b) Epi_DT 30

Figure1.12Ramachandran plot of ϕ and ψ dihedral angles of Epi_DT 32

Figure 1.13 Overall structure of Epi_DT 35

Figure 1.14Structure-based amino acid sequence alignment 36

Figure1.15Superimposedstructures of epimerases 38

Figure1.16Map of temperature factor of epimerases 39

Figure 1.17Surface map of catalytic center in Apo and lactose-Epi_DT structure 41

Figure1.18Overall structure of Epi_DT with its ligand 43

Figure1.19Interactions between ligand and important residues 44

Figure1.20Interaction between Epi_DT β1 strand and non-reducing sugar region 45

Figure1.21Conserved important residues interact with reducing sugar region in the

active sites of Epi_DT and cellobiose 2-epimerase from Rhodothermus marinus .46

Figure1.22Proposed catalytic reactions of Epi_DT 49

Figure1.23Proposed catalytic intermediates by Epi_DT 50

Figure1.24Ring opening process 51

Figure1.25Proposed epimerization mechanism 52

Figure1.26His247, Glu250, and His188 residues involved in the isomerization .53

Figure1.27Key residues in inter-conversion of lactose to epilactose and lactulose 54

Figure2.1Overall structure of XoMCAT 60

Figure2.2Multiple sequence alignment of MCAT structures 61

Figure2.3Protein-protein docking 66

Figure3.1Typical antibiotics having a macrolide ring 69

Figure3.2General aspects of the P450 catalytic cycle 70

Figure3.3Crystals of Apo-CYP107W1 and Oligomycin A-CYP107W1 72

Figure3.4Ramachandran plot of ϕ and ψ dihedral angles of Apo-CYP107W1 74

Figure3.5Overall structure of Apo-CYP107W1 77

Figure3.6Structure-based amino acid sequence alignment 78

Figure3.7Superimposed structures of P450s 79

Figure3.8Superimposedstructures of apo and complexedCYP107W1 81

Figure3.9Interactions between Heme and oligomycin A 82

FigureS.1Initial and optimized single crystals of CjPDF 104

FigureS.2.1Crystal of AbALR with nine different shapes 108

FigureS.2.2AbALR crystals having adequate dimensions 109

FigureS.3Initial ADH crystals obtained after three days 113

FigureS.4Crystals of XometB 117

FigureS.5Crystals of SFC-1 121

FigureS.6Optimized XoGroEL crystal 125

FigureS.7Crystals of chaperonine enzyme from X oryzae pv oryzae 130

FigureS.8A single AbDdl crystal 134

Abstract

Structure and Mechanism Studies of Sugar

Epimerase from Dictyoglomus turgidum

Pham, Tan Viet Department of Advanced Technology Fusion Graduate School of Konkuk University

Cellobiose 2-epimerase from Dictyoglomus turgidum(Epi_DT) is an enzyme

which inter-convertscellobiose to 4-O-β-D-glucopyranosyl-D-mannose and 4-O-β-Dglucopyranosyl-D-fructose and lactose to epilactose and lactulose.Idetermined the Epi_DT structure in apo and complex forms The three-dimensional structure of apo Epi_DT was determinedat 1.8 Å resolution and the complex structure of E250A mutated enzyme with lactose was determined at 2.0 Å resolution, whichshowed an open form of glucose (reducing part) in the catalytic pocket and was bound by 3 important histidine residues (His188, His247, and His377) The structural comparision with other epimerases suggested that His377 and Tyr114 were responsible for the ring opening process His247, His377, and His188 were involved in epimerization reaction based on an acid/base conversion catalysis mechanism His247, His188, and Glu250 were involved in the following isomerization reaction This study provides the useful information how Epi_DT inter-convertssubstrate sugars to useful industrial products such as lactulose and tagatose The crystal structure of Malonyl CoA-Acyl Carrier Protein

-Transacylase(XoMCAT) enzyme, encoded by the gene fabD (Xoo0880) from

Xoo, was determined at a resolution of 2.3Å in complex with a buffer ligand, N-cyclohexyl-2-aminoethansulfonic acid MCAT transfers malonyl group from malonyl-CoA to acyl carrier protein The transacylation step is essential

in fatty acid synthesis and XoMCAT is a putative target to develop antibacterial agents against Xoo CYP107W1 from Streptomyces avermitilis

catalyses the conversion of the macrolide oligomycin C (12-deoxyoligomycin A) to oligomycin A through a hydroxylation step at C12 I determined the CYP107W1 structure in apo and complex forms The three-dimensional structure of apo CYP107W1 was determined at 2.1 Å resolution and the complex structure of CYP107W1 with oligomycin A was determined at 2.6 Å resolution, which showed

an oligomycin A in the substrate binding pocket

Key words: Dictyoglomus turgidum, cellobiose 2-epimerase, lactose, carbonhydrate.Xanthomonas oryzae pv oryzae,bacterial blight (BB)

disease,XoMCAT, acyl carrier protein, Streptomyces avermitilis, CYP107W1,

oligomycin

Chapter 1 Structure and mechanism studies of sugar

epimerase from Dictyoglomus turgidum

1.1 Introduction

1.1.1 The important roles of carbohydrate in life

Carbohydrates form the most abundant group of natural products and are found in all classes of living organisms[1] They serve as a direct link between the energy of the sun and themetabolic energy that is required to sustain life In organisms capable of photosynthesis, solar energy is harvested to drive reactions in which glucose is synthesized from carbon dioxide and water The energy stored in this “carbon fixation” process then gradually moves upward into the food chain The living organisms that take the products of photosynthesis obtain useful energy

by oxidizing the carbohydrates back into carbon dioxide and water through the processes of glycolysis and respiration The carbohydrates that are most frequently used as metabolic vehicles are glucose, fructose, sucrose, lactose, and starch

In addition to their pivotal role in metabolism, carbohydrates also play an important structural role in many organisms Some examples of the latter type include cellulose, chitin, lipopolysaccharide, and the bacterial murein, all of which are derived from repeating sugar units which may have additional cross-linking components for rigidity Furthermore, many bioactive secondary metabolites such

as cardio-glycosides, macrolide antibiotics, and aminoglycoside antibiotics rely on

these sugar components for solubility and activity In addition, carbohydrates are used as convenient precursors for the biosynthesis of other important building blocks such as aromatic amino acids Carbohydrates also have many applications in industrial processes For example, the food industry uses sucrose as a sweetening agent, a preservative, and a raw material for fermentation Starch is used as a raw material for the manufacture of many goods Cotton is still one of the most popular fabrics and an important raw material for the textile industry Paper and other derivatives of cellulose are important for the manufacture of packaging materials and plastics

Figure 1.1 Major pathways in carbohydrate metabolism[2]

More recently, carbohydrates have been the focus of growing attention among biological molecules due to an increased recognition of their vital roles in many physiological processes[3] The diversity of structures that is made possible by carbohydrate building blocks is greater than that of oligonucleotides or oligopeptides[4].As an example, the number of all linear and branched isomers of a hexasaccharide is calculated to be over 1 × 1012 Even a simple disaccharide composed of two glucose units can be represented by 11 different structures In addition, nature has further enhanced the structural variations in carbohydrates by creating modified/unusual sugars such as deoxy-sugars, amino-sugars, and branched-chain sugars Such modifications can greatly influence thehydrophobicity and theoverall topologyof the glycosylated macromolecules[5]

Both normal and unusual sugars play important roles in cellular adhesion and cell-cell recognition, fertilization, protein folding, neurobiology, xenotransplantation, and target recognition in the immune response For example, glycosylation is a major posttranslational modification of membrane and secreted proteins in eukaryotic cells Alterations in the structures of glycans are associated with a variety of diseases, including metastatic cancer Similarly, glycol-sphingolipids, which contain oligosaccharides, are present in the plasma membrane, Golgi bodies, endosomes, and neuronal and synaptic membranes[6] Changes in the cell-surface expression profile of these compounds are associated with processes such as development, differentiation, organ regeneration, and oncogenic transformation Another example of the biological importance of carbohydrates is theposttranslational attachment of glycosylated phosphatidylinositol (GPI) anchors

to a wide variety of proteins found on the exterior surface of the eukaryotic plasma membrane GPI anchors have also been implicated as a sorting and targeting signal that marks the modified proteins for transport to the cell surface There is evidence for the involvement of GPI anchors in the activation of tyrosine kinases of the Src-kinase family These molecules may also serve as second messengers as well Therefore, it is clear that carbohydrates are not onlycritical for the storage and production of energy, but also intricately involved in many recognition and signaling events Given their biological significance, it is not surprising that alarge number of enzymes participate in the metabolism of carbohydrates[5]

1.1.2 Epimerization reaction mechanism

Many enzymatic transformations of carbohydrates involve the inversion of configuration at one or more stereogenic centers Such an epimerization has been commonly used in the conversion of D-sugars to the corresponding L-sugars It is also a convenient mechanism for accessing the structural diversity derived from a handful of common sugar precursors The reactions of most epimerases take place

at a chiral carbon adjacent to an activating moiety such as a carbonyl group, and the catalysis typically involves a simple deprotonation-reprotonation mechanism For example, enzymes such as the hexose 3,5-epimerases and hexose 5-epimerases use a 4-hexulose as the substrate, where the presence of a keto group at C4 facilitates the epimerization by lowering the pKa of the adjacent chiral center(s) However, the four reactions discussed in this section stand apart due to the fact that their catalyses involve epimerization at unactivated centers The C4 epimerization

of UDP-glucose to UDP-galactose mediated by UDP-galactose 4-epimerase is the best studied enzyme-catalyzed stereoinversion The second reaction, catalyzed by ribulose-5-phosphate 4-epimerase, also involves C4 epimerization, albeit via a different mechanism The last two examples are the interconversion of UDP-N-acetylmannosamine and UDP-N-acetylglucosamine and the transformation between CDP-paratose and CDP-tyvelose catalyzed by the corresponding 2-epimerases Again, bothcases involve the inversion of stereochemistry at the unactivated C2; however, their mechanisms are distinctly different As will be evident in the following sections, nature has clearly evolved a diverse range of catalysis to accomplish epimerization at chemically inert centers[5]

Enzymatic epimerization is an important modification for carbohydrates to acquire diverse functions attributable to their stereoisomers Epimerases and racemases are isomerases that catalyze inversion of the configuration around an asymmetric center of substrates Changing the stereochemistry of the hydroxyl substituent has biological significance in all branches of life In humans, mutations found in the UDP-glucose epimerase gene would be lethal In plant D-ribulose-5-phosphate 3-epimerase is a key enzyme in the Calvin cycle and the oxidative pentose phosphate pathway In pathogenic bacteria, epimerases are involved in the production of complex carbohydrate polymers used in their cell walls and envelopes

to protect them from the host immune system

Figure 1.2 Epimerase mechanism in general Mechanism A is based on the

assumption that this enzyme is NAD+ dependent This proposal involves the transient oxidation at C3 of 1 mediated by the NAD+ cofactor, deprotonation at C2 followed by reprotonation from the opposite side, and the final reduction of the C3 keto group to give 2 and regenerate the cofactor In mechanism B, the substrate 1 undergoes an anti-elimination of UDP from UDP-GlcNAc to form a 2-acetamidoglucal intermediate 2 A subsequent rebound of UDP in a synaddition could form the product UDP-ManNAc 2 Elements from these two mechanisms can

be incorporated into a hybrid mechanism (mechanism C) in which a tightly bound NAD+ is also a prerequisite[5]

Cellobiose 2-epimerase (CE) (EC 5.1.3.11), which was first found in the

culture fluid of an anaerobic ruminal bacterium, Ruminococcus albus[7], catalyzes

reversible epimerization of the D-glucose residue at the reducing end of oligosaccharides linked by β-1,4-glycosidic linkages, such as cellobiose, lactose, and 4-O-β-D-mannosyl-D-glucose to a D-mannose residue Although this is the sole enzyme responsible for catalyzing epimerization of the 2'-OH group of non-modified oligosaccharides, it has been shown to be widely distributed not only in

anaerobes but also in aerobes[8] In R albus and Bacteroides fragilis, the CE gene

comprises an operon together with the β-1,4-mannanase gene and the mannosyl-glucose phosphorylase gene encoding the enzyme catalyzing specific phosphorolysis of 4-O-β-D-mannosyl-D-glucose to α-D-mannose 1-phosphate and D-glucose Thus, CE may have a role in the metabolism of mannan; i.e., mannobiose produced by hydrolysis of mannan with β-1,4-mannanase is epimerized

4-O-β-to 4-O-β-D-mannosyl-D-glucose by CE, and then the epimerized product is phosphorylated by the phosphorylase[9] CE is an attractive enzyme to produce epilactose (4-O-β-D-galactosyl-D-mannose) from lactose Epilactose is a non-digestible oligosaccharide, and enhances proliferation of bifidobacilli and lactobacilli in the gut[10] This stimulation of growth of beneficial bacteria suppresses the conversion of primary bile acid to secondary bile acid, which is considered as a risk factor for colon cancer, and enhances absorption of minerals,including calcium, magnesium, and zinc[11] Furthermore, epilactose increases intestinal absorption of calcium through the paracellular route[12].For application of epilactose as a functional foodstuff, practical methods for preparation

of epilactose have been developed[13]

Figure 1.3 Proposed catalytic mechanisms for AGE and YihS In the case of

AGE, the place of the resonance structure of N-acetyl group is surrounded by a dotted circle Thin and bold arrows exhibit forward and reverse reactions, respectively[14]

CE enzymes belong to the N-acetyl-D-glucosamine 2-epimerase (AGE) superfamily In this superfamily, the structures of two epimerases, i.e., AGE and aldose–ketose isomerase YihS have been reported, and their catalytic mechanisms have been suggested [15] The three-dimensional structures of AGEs from

Anabaena sp CH1 (PDB ID: 2GZ6)[14], and porcine kidney (PDB ID: 1FP3)

[16]have been determined, and the catalytic mechanism of AGE was postulated based on the results of structural and site-directed mutational studies[14].In the case

of AGE, two histidines, His239 and His372 (numbered with reference to ɑAGE) at the catalytic center surrounded by inner helices, were responsible for general acid/base catalysis to achieve interconversion between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc) These two histidines interacted electrostatically with specific glutamates to stabilize the positive charge

of their imidazole rings Furthermore, the structure of YihS, which catalyzes isomerization of an unmodified sugar, has been also determined The structures of

YihS from Escherichia coli (EcYihS) (PDB ID: 2RGK) and Salmonella enterica

(SeYihS) (PDB ID: 2AFA), which show high levels of activity toward mannose and glucose, are similar to that of AGE Although RaCE shows sequence similarity with low identity to AGE and YihS, several amino acids in the catalytic centers of AGE and YihS are well conserved in CE (Arg52, His243,Glu246, Trp249, Trp304, Glu308, and His374 inRɑCE), and mutational experiments with RɑCE indicated that these residues are critical for the catalytic activity[17] However, the substrate specificities of CE, AGE, and YihS differ from each other in terms of substrate chain length and in the chemical group at the C2 position of the substrate While AGE reacts with modified sugars, CE and YihS react with unmodified sugars Moreover, epimerization by AGE and YihS is specific to monosaccharides, whereas

CE reacts with oligosaccharides Although the common epimerization reaction occurring on deprotonation from a chiral carbon was proposed for both AGE and YihS, the deprotonation of unmodified sugars such as in YihS is still unclear

1.1.3 Three-dimensional structure determination methods

Three-dimensional structure allows us to understand biological processes at the most basic level: which molecules interact, how they interact, how enzymes catalyze reactions, how drugs act In some cases, it can allow us to understand disease at an atomic level, such as the stickling of red blood cells We can also exploit 3D structure in developing new drugs There are a number of methods for studying 3D structure such as crystallography, electron microscopy, atomic force microscopy, nuclear magnetic resonance, etc

1.1.3.1 Electron microscopy

An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than visible light (photons), and can achieve better than 50 pm resolution [18] and magnifications of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000

The electron microscope uses electrostaticand electromagnetic"lenses" to control the electron beam and focus it to form an image These lenses are analogous

to, but different from the glass lenses of an optical microscope that forms a magnified image by focusing light on or through the specimen (Fig 1.4) In transmission, the electron beam is first diffracted by the specimen, and then, the

electron microscope “lenses" re-focus the beam into a Fourier-transformedimage of the diffraction pattern for the selected area of investigation The real image thus formed is magnified by a factor ranging from a few hundred to many hundred thousand times, and can be viewed on a detecting screen or recorded using photographic film or plates or with a digital camera

Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals Industrially, the electron microscope is primarily used for quality control and failure analysis in semiconductor device fabrication

apparatus(http://en.wikipedia.org/wiki/Transmission_electron_microscope)

The advantages of electron microscopy are that the specimen needs not to be

a single crystal or even a polycrystalline powder; the result can be seen directly via eye or camera

The major disadvantage of the transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers Biological specimens typically require chemically fixed, dehydrated and embedded

in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning Sections of biological specimens, organic polymers and similar materials may require special `staining' with heavy atom labels in order to achieve the required image contrast

1.1.3.2 Atomic force microscopy

Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit The AFM is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale

The advantages of AFM are that it provides a three-dimensional surface profile; samples viewed by AFM do not require any special treatments that would irreversibly change or damage the sample, and does not typically suffer from charging artifacts in the final image; most AFM modes can work perfectly well in ambient air or even a liquid environment This makes it possible to study biological macromolecules and even living organisms

The disadvantages of AFM are that it can only image limited size of object, slow scanning speed,the possibility of image artifacts

1.1.3.3 Nuclear magnetic resonance (NMR)

Nuclear magnetic resonance (NMR) spectroscopy is one of the methods available for three-dimensional structure determination of proteins and nucleic acids

at atomic resolution, since the NMR data can be recorded in solution Considering that body fluids such as blood, stomach liquid and saliva are protein solutions where these molecules perform their physiological functions, knowledge of the molecular structures in solution is highly relevant In the NMR experiments, solution conditions such as the temperature, pH and salt concentration can be adjusted so as

to closely mimic a given physiological fluid Conversely, the solutions may also be changed to quite extreme non-physiological conditions, for example, for studies of protein denaturation Furthermore, in addition to protein structure determination, NMR applications include investigations of dynamic features of the molecular structures, as well as studies of structural, thermodynamic and kinetic aspects of interactions between proteins and other solution components, which may either beother macromolecules or low molecular weight ligands Again, for these supplemental data it is of keen interest that they can be measured directly in solution.The most disadvantage of NMR is that it is not for the availability of higher molecular masses, the highest molecular mass which was examined successfully is just a 64kDa protein-complex Moreover, the resolution of determined structure is usually low

1.1.3.4 X-ray crystallography

X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions From the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture

of the density of electrons within the crystal From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information

The technique of single-crystal X-ray crystallography has three basic steps as followed:

 The first- and often most difficult-step is to obtain an adequate crystal of the protein The crystal should be sufficiently large (typically larger than 0.1 mm

in all dimensions), pure in composition and regular in structure, with no significant internal imperfections such as cracks or twinning

 In the second step, the crystal is placed in an intense beam of X-rays, usually

of a single wavelength (monochromatic X-rays), producing the regular pattern

of reflections As the crystal is gradually rotated, previous reflections disappear and new ones appear; the intensity of every spot is recorded at every orientation of the crystal Multiple data sets may have to be collected, with each set covering slightly more than half a full rotation of the crystal and typically containing tens of thousands of reflections This step builds a map of electron density

 In the third step, these data are combined computationally with complementary chemical information to produce and refine a model of the arrangement of atoms within the crystal The final, refined model of the atomic arrangement-now called a crystal structure-is usually stored in a public database

Figure1.5 Workflow for solving the protein structure by X-ray

crystallography (http://en.wikipedia.org/wiki/X-ray_crystallography)

1.1.3.4.1 Protein crystallization principle

In the X-ray structural analysis of a protein, obtaining suitable single crystals

is also the least understood step Protein crystallization is mainly a trial-and-error procedure in which the protein is slowly precipitated from its solution As a general rule, the purer the protein is, the better the chances to grow crystals

The crystallization of proteins involves four important things: purity of the protein; suitable solvent, a salt or an organic compound to dissolve the protein; forming nuclei for crystal growth at a high supersaturation and crystal growth when nuclei have formed Among those, crystallization proceeds in two phases: nucleation and growth To easily design crystallization experiments, a crystallization phase diagram was created to show which state liquid, crystalline or amorphous solid (precipitate) is stable under a variety of crystallization parameters[19]

The phase diagram is obtained experimentally by varying two parameters at a time, thus representing a two-dimensional ‘slice’ of the multidimensional space of parameters relevant to crystallization In a typical crystallization phase diagram, it is distinguished between four areas: an area of very high supersaturation, where the protein will precipitate; an area of moderate supersaturation, where spontaneous nucleation will take place; an area of lower supersaturation just below the nucleation zone, where crystals are stable and may grow, but no further nucleation will take place (referred to as the metastable zone, this area is thought to contain the best conditions for growth of large, well-ordered crystals); and an undersaturated area, where the protein is fully dissolved and will never crystallize [19, 20]

Techniques have been used for crystallization settings such as batch crystallization (the precipitating reagent is instantaneously added to a protein solution, suddenly bringing the solution to a state of high supersaturation), free interface diffusion (the protein solution and the solution containing the precipitant are layered on top of each other in a small-bore capillary), vapor diffusion (most widely used method in which the protein solution is either a hanging or sitting drop that equilibrates against a reservoir containing crystallizing agents), dialysis (dialysis membrane containing protein against precipitant solution or capillary)

Figure1.6.Schematic illustration of a protein crystallization phase diagram[20]

Adjustable parameters include precipitant or additive concentration, pH and temperature The four major crystallization methods are represented: (i) microbatch, (ii) vapor diffusion, (iii) dialysis and (iv) free interface diffusion (FID) Each

involves a different route to reach the nucleation and metastable zones, assuming the adjustable parameter is precipitant concentration The filled black circles represent the starting conditions Two alternative starting points are shown for FID and dialysis because the undersaturated protein solution can contain either protein alone or protein mixed with a low concentration of the precipitating agents The solubility is defined as the concentration of protein in the solute that is in equilibrium with crystals The supersolubility curve is defined as the line separating conditions under which spontaneous nucleation (or phase separation or precipitation) occurs from those under which the crystallization solution remains clear if left undisturbed This figure was taken from Figure 1 of reference[20]

1.1.3.4.2 Vapor diffusion methods for protein crystallization

There are at least seven practical methods used for macromolecule crystallization including micro-batch experiment, vapor diffusion, bulk crystallization, free interface diffusion, dialysis, temperature-induced, and seeding Among these methods, vapor diffusion is the most widely used methods of crystallization

Figure 1.7 Crystalsin the reservoir solution of (A) hanging drop protein

crystallization experiments or in (B) sitting drop protein crystallization setups (www.emeraldbiosystems.com by Peter Nollert)

Vapor diffusion methods includes: hanging drop, sitting drop, sandwich, and capillary methods The most popular protocols are hanging drop and sitting drop Both entail a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a large reservoir containing similar buffer and precipitant in higher concentrations Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete

1.2.Methods

1.2.1 Cloning of cellobiose 2-epimerase

The genomic DNA from Dictyoglomus turgidum was extracted using a

genomic DNA extraction kit (Qiagen, Hilden, Germany) The gene (1,173 bp) encoding for a putative N-acyl-D-glucosamine 2-epimerase (Epi_DT) was

amplified by PCR using the genomic DNA isolated from D turgidum as a template

The sequences of the oligonucleotide primers used for gene cloning were based on

the DNA sequence of D turgidum N-acyl-D-glucosamine 2-epimerase (GenBank

accession number, NC_011661.1) Forward

(5′-AACATATGGATTTAAAAGTTTTAAAAAGTG-3’) and reverse primers (5′- TTCTCGAGTTAAATCCTTTTTATTACCTCAAGA-3′) were designed to

introduce the NdeI and XhoI restriction sites (underlines), respectively The

PCR-amplified DNA fragments were purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and inserted into a pET-28a vector digested with the

same restriction enzymes The resulting plasmid was transformed into E coli

ER2566 and plated on Luria-Bertani (LB) agar containing kanamycin (50 μgml-1) A kanamycin-resistant colony was selected, and plasmid DNA from the transformant was isolated using a plasmid purification kit (Promega, Madison, WI, USA)

I sub-cloned the DT_Epimerase gene from the pET-28a-Epi_DT vector into a

pET-29b-His-Tev vector by PCR using the forward (5’-CCC CCC ATA TGG ATT TAA AAG TTT TAA AAA GTG-3’) and reverse (5′-CCC CCC CCA TGG TTA

AAT CCT TTT TAT TAC CTC-3′) primers having NdeI and BamHI restriction sites

The amplified gene was inserted into the expression vector pET-29b-His-Tev which

was modified to have seven histidine residues and TEV cleavage site at the

N-terminus of the gene product from the original pET-29b vector (Novagen) in order

to facilitate protein purification The resulting plasmid was transformed into E coli

NEB Turbo (New England Biolabs) and plated on LB agar containing kanamycin

(50 μgml-1) A kanamycin–resistant colony was selected and plasmid DNA from the

transformant was isolated using plasmid purification kit (FavorPrepTM, Favorgen,

Taiwan) All DNA sequencing was carried out at Macrogen company (Seoul, Korea)

H188A, H247A, H377A, and E250A mutants were prepared by using

Muta-direct Site Directed Mutagenesis Kit (Intron Biotechnology) in accordance with the

manufacturer’s protocol The sequence of mutant clones were confirmed by DNA

sequencing was carried out at Macrogen company (Seoul, Korea)

Figure 1.8 pET-29b-His-Tev vector sequence which was made by modifying

pET-29b vector to have seven histidine residues and a TEV cleavage site at the terminus of the gene product in order to facilitate protein purification

N-1.2.2 Overexpression and Purification of cellobiose 2-epimerase

The pET28a and pET29b-His-Tev expression vectors containing the coding

sequence for Epi_DT gene were introduced into Escherichia coli BL21(DE3) cells

Cells were grown at 288 K until the OD600 reached to 0.6 in LB medium containing50µ g ml-1 kanamycin Protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG)to a final concentration of 0.5mM and the cells were cultured at the same temperature for 16h more Cultured cells were harvested by centrifugation for 30min at 6,000× g (Vision VS24-SMTi V5006A rotor) at 277 K and then re-suspended in 25mM Tris/HCl pH 7.5, 300mM NaCl, 10mM imidazole on ice A sonicator was used to discrupt the re-suspended cells (Sonomasher, S&T Science, Korea) The crude lysate was centrifuged for 30 min at 21,000× g (Vision VS24-SMTi V508A rotor) at 277 K An SDS-PAGE experiment indicated that 10% of total expressed Epi_DTwas soluble The supernatant containing soluble Epi_DT protein was applied into Ni-NTA resin (Novagen) and affinity purification was performed based on the manufacturer’s protocol at 277 K The elution buffer containing 250mM imidazole was used to elute the 7xHis-tagged Epi_DT The elution was dialyzed for 8 h at 277 Kagainst dialysis buffer [25mM Tris/HCl pH 7.5, 15mM NaCl] 100 mg Epi_DT was purified from 4 l of culture, totally The 7xHis tag was removed by treating with TEV protease overnight at 277K Further purification was carried out on an UNO 6Q column (Biorad) The homogeneity of the purified protein was checked via SDS-PAGE The purified protein was concentrated to 7.5mg ml-1 which used in the crystallization process

After purification, the homogeneity of the purified Epi_DT was confirmed by

10% SDS-PAGE gel (Fig 1.9)

Figure 1.9 Purified His-tagged Epi_DT and untagged Epi_DT are shown on

a 10% SDS-PAGE gel Lane M1, His-tagged Epi_DT (~48kDa) Lane M2, untagged Epi_DT (~ 46kDa) Lane P, prestained protein ladder (Fermentas).The mutated enzymes were purified by using the same vector which could cut the 7-His tag These enzyme purifications base on the protocol which used to purify untagged Epi_DT

1.2.3 Crystallization and X-ray data collection

The sitting-drop vapor–diffusion method was used for initial crystallization at

287 K in 96-well Intelli plates (Art Robbins) by a Hydra II e-drop automated pipetting system (Matrix) and screening kits from Hampton Research (Index, Crystal Screen, Crystal Screen Cryo, Crystal Lite and PEGRx), Emerald Biosystems (Wizard, Wizard precipitant synergy) and Molecular Dimensions (MorpheusTM MD1-46 kit) Two kinds of Epi_DT (with and without 7xHis-tag) at 7.5mg/ml of concentration were set up for crystallization 0.5 µl solution of protein was mixed with 0.5 µl reservoir solution and equilibrated against 50 µl reservoir solution After two days, crystals of His-tagged Epi_DTprotein were observed inthe condition G10 of Index, Hampton Research [25%(w/v) Polyethylene glycol 3,350, 0.2M magnesium chloride hexahydrate, 0.1M Bis-Tris pH 5.5] This condition was used to reproduce and optimize crystals in the hanging drops method where drops contained 0.9 μl of protein solution mixed with 0.9 μl of reservoir solution, and where the drops were equilibrated against 1 ml of reservoir solution Optimization was achieved by varying concentration of PEG 3,350 (w/v) (22% - 27%) and pH of 0.1M Bis-Tris (4.5-6.0) Tetragonal shapedcrystals of His-tagged Epi_DTwere obtained after one week using a reservoir solution containing 23%(w/v) PEG 3350, 0.2M magnesium chloride hexa-hydrate, 0.1M Bis-Tris pH6.0 The fully grown crystals (0.05 × 0.10 × 0.02 mm) were flash-cooled at 100 K in liquid nitrogen using 20%(v/v) 2-Methyl-2,4-pentanediol, 23%(w/v) PEG 3350, 0.2M magnesium chloride hexa-hydrate, 0.1M Bis-Tris pH6.0

Because of the data which collected from His-tagged Epi_DT was not good enough to solve the structure, we changed the expression vector and the pET-29b-His-Tev was used as the new expression vector The Epi_DT was purified by using pET-29-His-Tev was treated with TEV protease to cut 7-His tag Initial crystals of untagged Epi_DT was obtained after seven days using a reservoir solution containing 26.8%(v/v) Isopropanol, 10.05%(w/v) PEG 8000, 0.1M

Imidazole/Hydrochloric acid pH 6.5 (Fig 1.10c), and optimized crystal of

Epi_DTwas obtained using 26.0%(v/v) Isopropanol, 10.0%(w/v) PEG 8000, 0.1M

Imidazole/Hydrochloric acid pH 7.0 (Fig 1.10d)

X-ray diffraction data were collected from the cryoprotected crystals at the beamline 5C SBII of Pohang Light Source (PLS), South Korea, and at beamline XU32 of Spring-8, Riken, Japan Crystals diffracted to 2.6Å (His-tagged) and 1.8Å (untagged) resolution, respectively The diffraction data were integrated and scaled using DENZO and SCALEPACK, respectively [21]

His-tagged Epi_DT crystals belong to the crystallographic space group P21 The unit-cell parameters are a = 63.9 Å , b = 85.1 Å , c = 79.8 Å , and β = 110.8º The

diffraction image showed a 2.85 Å resolution circle (Fig 1.11a) The space group

was assigned by auto-indexing [22] and data-collection statistics are provided in table 1.1 According to the Matthews coefficient calculation [23], there are

probably two molecules in the asymmetric unit, corresponding to a VM of 2.18

Å3 Da-1 and solvent content of 43.74% Molrep in CCP4 program package [24] with

N-acetylglucosamine 2-epimerase from Xylella fastidiosa (PDB ID: 3GT5, 22%

sequence identity) as a search model was successful and showed dimers in the

asymmetric unit Although the initial R-factor from the molecular replacement search was 55.0%, the resulting electron density maps were clear and no clashes were found between molecules After rigid body and first restrained refinement by Refmac5 [25], the R-factor decreased to 31.7% and the R-free was 44.8%

Untagged Epi_DT crystal belongs to the crystallographic space group P212121 The unit cell parametersare a = 55.9 Å , b = 80.0 Å , and c = 93.7 Å The diffraction

image showed a 2.30 resolution circle (Fig 1.11b) The space group was assigned

by auto-indexing [22] and data-collection statistics are provided in Table 1.1 According to the Matthews coefficient calculation [23], there is one molecule in the

asymmetric unit, corresponding to a VM of 2.26 Å3 Da-1 and solvent content of 45.59% Molrep in CCP4 program package [24] with the same search model was successful and showed a monomer in the asymmetric unit The initial R-factor from the molecular replacement search was 48.3%, and the resulting electron density maps were of high quality and no clashes were found between molecules After rigid body and first restrained refinement by Refmac5 [25], the R-factor decreased

to 24.7% and the R-free was 27.8%

Mutated Epi_DT was co-crystallized with cellulose, maltose, and lactose in 1:10 ratio of molecular Then I got the crystal with lactose in the same

crystallization buffer condition as Untagged Epi_DT form (Fig 1.10e) This crystal

belongs to the crystallographic space group P212121 The unit cell parametersare a = 55.5 Å , b = 79.7 Å , and c = 90.0 Å at 2.05 Å resolution, respectively

Figure 1.10 Crystals of His-tagged and untagged Epi_DT (a) Initial crystal

obtained after three days of His-tagged Epi_DT (b) Optimized crystal of His-tagged Epi_DT (c) Initial crystal after 7 days of untagged Epi_DT (d) Optimized crystal of untagged Epi_DT (e) Complex crystal of E250A mutated Epi_DT with α-lactose

(b) (a)

(e)